Emphasis is given to develop a fast, direct, and safe assay (no radioactivity) to determine binding characteristics of nucleic acid binding proteins to nucleic acids. The method makes use of spin probes which are covalently bound to nucleic acid homopolymers at random intervals. The spin labeling of the homopolymers is carried out according to a well established procedure developed in this laboratory. The binding event is directly monitored by electron spin resonance spectroscopy on small sample quantities without tedious sample manipulations which possibly can partially denature the proteins. The fraction, f, of the spin labeled nucleic acid in its complexed state will be obtained by minimizing the sum of the absolute difference between the calculated and observed spectra at each magnetic field value. In conjunction with the labeled homopolymers unlabeled nucleic acids of various sources are used for competition experiments and for determining the rate of transfer of proteins from labeled to unlabeled nucleic acids. It should be stressed that one of the unique features of this assay lies in its ability to directly follow the fate of a particular nucleic acid in a complex multiequilibria system. The nucleic acid binding proteins to be used in the assay are: gene 32 protein (P32), gene 5 protein (P5), and leukoviral RNA-directed DNA polymerase (reverse transcriptase). Because the assay is fast and requires little material it will also be helpful for the determination of binding characteristics of other nucleic acid binding proteins.